The present invention relates to a method of genotyping CYP2A6 gene, for diagnosing a metabolic ability of a substance which is to be a substrate of human cytochrome P4502A6 (hereinafter abbreviated as CYP2A6) and a diagnostic kit.
There exists genetic polymorphism in CYP2A6, which has been shown to cause individual differences of the metabolic abilities of the substances which are to be substrates [Japanese Patent Laid-Open No. Hei 9-187300 and J. Pharmacol. Exp. Trer. 1999, 289(1): 437-442]. A PAF (platlet-activating factor) receptor antagonist (+)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one (SM-12502) is a representative substrate for CYP2A6, and it has been known that an in vivo metabolic ability of SM-12502 is low in an individual having whole deletion-type mutation of CYP2A6 gene [Japanese Patent Laid-Open No. Hei 9-187300 and J. Pharmacol. Exp. Trer. 1999, 289(1): 437-442]. Therefore, it is thought to be very useful that a patient is diagnosed for his/her genotype to previously know the metabolic ability, before administering a drug which is to be a substrate for CYP2A6, such as SM- 12502, from the viewpoint of subjecting the patient to an appropriate treatment in accordance with the individual differences of the patient while preventing adverse action.
From the viewpoints described above, the present inventors have so far studied on a method of determining whole deletion-type mutation of CYP2A6 gene. Japanese Patent Laid-Open No. Hei 9-187300 and J. Pharmacol. Exp. Trer. 1999, 289(1): 437-442 have suggested deletions of exons 1 to 9 of CYP2A6 gene. Thereafter, by elucidation of the structure of this deletion-type gene, there has been developed a diagnostic method for determining a homozygous deletion individual (the term xe2x80x9chomozygous deletionxe2x80x9d refers to deletion of both alleles), only summary of the diagnostic method being described in Pharmaceutical Res., 15(4), 517-524, 1998. However, in the diagnosis by this diagnostic method, there arises an unexpected problem that the frequency of a heterozygous deletion individual (the term xe2x80x9cheterozygous deletionxe2x80x9d refers to deletion of one of alleles) is far higher than a theoretically calculated frequency.
Accordingly, an object of the present invention is to provide a genotyping method of human CYP2A6 gene, for diagnosing a metabolic ability of a substance which is to be a substrate for human CYP2A6 such as SM-12502 in a patient, a diagnostic kit and the like.
There is a possibility that enzymatic activity is also affected in a case where an individual heterozygously has a whole deletion-type mutation of CYP2A6 gene. Therefore, it is thought that a diagnostic method for accurately determining a heterozygous deletion individual as well as a homozygous deletion individual is necessary, from the viewpoint of giving a patient an appropriate treatment in accordance with individual differences of the patient. Therefore, the present inventors have made various studies on the causation why the frequency of the heterozygous deletion individual is far higher than the theoretically calculated frequency and the like. As a result, there was considered the existence of a new polymorphic gene not reported so far, so that CYP2A6 cDNA was cloned from a human liver sample determined as heterozygote by the above-mentioned old diagnostic method [Pharmaceutical Res., 15(4), 517-524, 1998], and its nucleotide sequence was analyzed. As a result, there was found a new polymorphic gene (hereinafter referred to as xe2x80x9cconversion-typexe2x80x9d) in which there are no mutations in the 5xe2x80x2-untranslational region and the coding region, and a region having a size of about 60 bp in the 3xe2x80x2-untranslational region is converted to a corresponding region of CYP2A7 gene. Here, the term xe2x80x9cCYP2A7 genexe2x80x9d refers to another gene of a CYP2A subfamily, which locates on the chromosome 19, the same position as that in CYP2A6 gene, and has high homology to CYP2A6 gene (Am. J. Hum. Genet. 57: 651-660, 1995).
There is a possibility that enzymatic activity is also affected in a case where an individual homozygously or heterozygously has a conversion-type mutation of CYP2A6 gene, which is newly found by the present inventors. Therefore, as a result of intensive studies, the present inventors have succeeded in establishing a new diagnostic method capable of genotyping CYP2A6 gene on the bases of the difference in the three kinds of the gene structures, wild-type, whole deletion-type and conversion-type, and the present invention has been perfected thereby.
Specifically, the present invention relates to:
(1) a method of determining a genotype of CYP2A6 gene on the basis of a difference in gene structures of a wild-type gene of human cytochrome P4502A6 (CYP2A6) and at least one kind of mutant-type gene associated with mutation of CYP2A6 gene;
(2) the method according to item (1) above, wherein at least one kind of mutation occurred in the mutant-type gene exists in exon 9 of human cytochrome P4502A7 (CYP2A7) gene, or exon 9 of CYP2A6 gene;
(3) the method according to item (1) above, wherein one kind of mutant-type gene associated with mutation of CYP2A6 gene is used, and the mutant-type gene is conversion-type CYP2A6 gene comprising the nucleotide sequence of SEQ ID NO: 4;
(4) the method according to item (1) above, wherein homozygous deletion and heterozygous deletion of CYP2A6 gene are determined by using at least two kinds of mutant-type genes associated with mutation of CYP2A6 gene;
(5) the method according to item (4) above, wherein the above-mentioned at least two kinds of mutant-type genes associated with mutation of CYP2A6 gene are whole deletion-type CYP2A6 gene comprising the nucleotide sequence of SEQ ID NO: 1 and conversion-type CYP2A6 gene comprising the nucleotide sequence of SEQ ID NO: 4;
(6) the method according to any one of items (1) to (5) above, characterized by analyzing the difference in gene structures by a method comprising PCR method and/or hybridization method;
(7) the method according to any one of items (1) to (6) above, wherein the method comprises the following steps:
(a) preparing a genomic DNA from a human tissue;
(b) preparing 5xe2x80x2-primer capable of hybridizing to both exon 8 of CYP2A6 gene and exon 8 of CYP2A7 gene, and 3xe2x80x2-primer capable of specifically hybridizing to exon 9 of CYP2A6 gene;
(c) carrying out amplification reaction by PCR using primers prepared in step (b) as a primer pair, with the genomic DNA prepared in step (a) as a template; and
(d) digesting the amplified product obtained in step (c) with a restriction enzyme, thereby detecting a restriction fragment length polymorphism;
(8) the method according to item (7) above, wherein the 5xe2x80x2-primer has the nucleotide sequence of SEQ ID NO: 5, and the 3xe2x80x2-primer has the nucleotide sequence of SEQ ID NO: 6;
(9) the method according to item (7) or (8) above, wherein the restriction enzyme is AccII or an isoschizomer thereof, and Eco81I or an isoschizomer thereof;
(10) the method according to item (6) above, wherein the method comprises analyzing the difference in gene structures of wild-type CYP2A6 gene and conversion-type CYP2A6 gene, with a DNA fragment having a length of at least 5 bp, the DNA fragment comprising at least one boundary region between CYP2A6 gene and CYP2A7 gene in the nucleotide sequence of SEQ ID NO: 4;
(11) the method according to item (10) above, wherein the method comprises the following steps:
(a) preparing a genomic DNA from a human tissue; and
(b) subjecting the genomic DNA prepared in step (a) or a fragment thereof to hybridization reaction with the DNA fragment having a length of at least 5 bp, the DNA fragment comprising at least one boundary region between CYP2A6 gene and CYP2A7 gene in the nucleotide sequence of SEQ ID NO: 4;
(12) the method according to item (10) or (11) above, characterized in that the DNA fragment is in the form bound to a solid support;
(13) a method of determining a genotype of a CYP gene, characterized by carrying out a method comprising the method of any one of items (1) to (12) above to determine the genotype of the CYP gene;
(14) a conversion-type CYP2A6 gene comprising the nucleotide sequence of SEQ ID NO: 4;
(15) a DNA fragment having a length of at least 5 bp, comprising at least one boundary region between CYP2A6 gene and CYP2A7 gene in the nucleotide sequence of SEQ ID NO: 4;
(16) a DNA fragment having a length of at least 60 bp, comprising two boundary regions between CYP2A6 gene and CYP2A7 gene in the nucleotide sequence of SEQ ID NO: 4;
(17) a solid support with which the DNA fragment of item (15) or (16) above is bound;
(18) a diagnostic kit for determining a genotype of a CYP gene, comprising the DNA fragment of item (15) or (16) above, or the solid support of item (17) above;
(19) a DNA fragment having the nucleotide sequence of SEQ ID NO: 5 or 6;
(20) a diagnostic kit for determining a genotype of CYP2A6 gene, comprising 5xe2x80x2-primer capable of hybridizing to both exon 8 of CYP2A6 gene and exon 8 of CYP2A7 gene; 3xe2x80x2-primer capable of specifically hybridizing to exon 9 of CYP2A6 gene; AccII or an isoschizomer thereof; and Eco81I or an isoschizomer thereof; and
(21) the diagnostic kit according to item (20) above, wherein the 5xe2x80x2-primer has the nucleotide sequence of SEQ ID NO: 5, and the 3xe2x80x2-primer has the nucleotide sequence of SEQ ID NO: 6.